DESCRIPTION (from applicant's abstract): The overall goal of the proposed research is to reveal how homologs and variants of ribonuclease A (RNase A) promote tumor cell death. Onconase, which is an amphibian homolog of RNase A, is now undergoing Phase III human clinical trials for the treatment of malignant mesothelioma and Phase I/II trials for the treatment of metastatic renal carcinoma. In contrast to onconase, RNase A is not cytotoxic. In the initial funding period, variants of RNase A were created that, like onconase, were able to evade the endogenous ribonuclease inhibitor protein (RI) that resides in the cytosol of mammalian cells. These variants were toxic to tumor cells. This finding portends the development of a new class of cancer chemotherapeutics based on homologs and variants of RNase A. The specific aims of the proposed research are: (1) to determine how RNase A homologs bind to the cell surface, (2) to discover the route taken by RNase A homologs to cytosolic RNA, (3) to reveal fundamental information about the subcellular localization and intracellular function of RI, and (4) to create a human homolog of RNase A that is more toxic to tumor cells than onconase. Relevant properties of RNase A homologs and variants will be assessed in comparison to onconase (positive control) and wild-type RNase A (negative control). These properties include cytotoxicity, RI evasion, ribonucleolytic activity, and conformational stability. Finally, the three-dimensional structures of RNase A homologs and variants with notable cytotoxicity will be determined by X-ray diffraction analysis. The proposed research is designed to reveal new insights into the basis of ribonuclease cytotoxicity, and could lead to new cancer chemotherapeutics.